Toxoplasma gondii is a common parasitic infection of humans that causes opportunistic disease in immunocompromised patients, including those with AIDS or those undergoing chemotherapy for cancer or organ transplant. The symptoms of infection vary considerably, ranging from subclinical to severe, although the factors that control these disparate outcomes are poorly understood. One component that likely contributes to disease severity is the genetic makeup of the parasite. However, little is known about the specific parasite genes that are responsible for influencing disease severity. Our previous studies have shown that T. gondii has a highly unusual genetic makeup consisting of three clonal lineages that are wide spread in North America and Europe. These three strain types differ substantially in the immune responses they evoke and the severity of infections that they cause. The proposed studies will investigate the molecular basis of acute virulence in Toxoplasma gondii using forward and reverse genetics to identify genes important for pathogenesis. We will explore the role of secretory kinases, recently implicated as major virulence determinants of T. gondii, and characterize their cellular and biochemical mechanisms of action. Additional genetic crosses between different parasite genotypes will be undertaken to map genes that regulate differences in pathogenesis, including genes that control tissue migration, induction of immune responses, and acute virulence. Finally, we will identify genes that contribute to pathogenesis of newly described T. gondii lineages from other geographic regions. Forward and reverse genetic analyses will be used to map genes and determine the molecular basis of virulence in these newly identified parasite lineages. These studies will provide important fundamental knowledge about the identity and mechanisms of action of virulence determinants in T. gondii that contribute to human disease. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is a widespread parasitic infection that can cause severe disease in immunocompromised patients. Our studies seek to define the factors that contribute to the severity of disease caused by this parasite. By understanding the virulence determinants of the parasite that enable it to cause harm, it may be possible to develop new therapeutic approaches to combat human infection. The findings are highly relevant to the pathology of toxoplasmosis in immunocompromised patient, including individuals with AIDS.